During the next project year we shall continue our hemepeptide studies and our chemical labeling studies of the heme pocket of P-450 hemeproteins with substrate and inhibitor derived affinity and photoaffinity labels. We propose to focus on cytochrome P-450CAM of P. putida as our model compound and on P-450LM-2 of rabbit liver microsomes to determine the applicability of our newly developed procedures and concepts to other P-450s. Amino acid sequences of BrCN derived hemepeptides of these two proteins shall be determined initially by Edman degradation of apopeptides immobilized on small glass beads. The results will be confirmed by additional cleavages with enzymes and specific chemical reagents. This work will furnish sequence information was well as identification of contact residues from affinity and photoaffinity labeled samples of these hemepeptides. By the use of multifunctional labels we hope to identify features of the folding pattern of the heme domain and to get a glimpse at the size and topology of the heme pocket. The relative positions and reactivities of sulfhydryl groups at the substrate binding site which are crucial for substrate binding and orientation relative to the heme iron will be determined by means of radiolabled substrate derivatives. The goal is the assignment of specific roles to individual sulfhydryl groups during the hydroxylation reaction. A strategy will be applied for differential labeling of SH groups by different radiolabeled reagents to render specific SH groups uniquely susceptible to proteolytic cleavage thereby generating large peptide fragments and pinpointing the position of essential SH groups within the linear sequence. Mercury derivatives of substrates and inhibitors will be developed to prepare heavy metal derivatives of P-450CAM for use in X-ray diffraction studies.